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Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae
Experimental Parasitology 135 (2013) 564–569
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Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae Simon Haarder ⇑, Per W. Kania, Qusay Z.M. Bahlool, Kurt Buchmann Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 7, Frederiksberg C, Denmark
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Oral challenge with live Anisakis
simplex induces expression of numerous immune relevant genes in rainbow trout. The CD8 and IgM genes were significantly down-regulated in infected fish at 1 and 8 days post infection (d.p.i.). A significant up-regulation was seen in the CD4 gene in fish which rejected the worms at 8 d.p.i. C3 and precerebellin genes were both significantly up-regulated in infected fish 4 d.p.i. The SAA gene exhibited significant up-regulation at several different sampling points.
a r t i c l e
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Article history: Received 31 January 2013 Received in revised form 19 August 2013 Accepted 6 September 2013 Available online 16 September 2013 Keywords: Anisakis simplex Fish immunology Gene expression Fish-nematode experimental model qPCR Rainbow trout
a b s t r a c t Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fishnematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8 days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; ‘‘worms rejected’’ (worms), ‘‘worms present’’ (+worms) and a combined group consisting of samples pooled from both previous groups (/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8 d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24 h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8 d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4 d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae. Ó 2013 Elsevier Inc. All rights reserved.
⇑ Corresponding author. E-mail addresses: [email protected] (S. Haarder), [email protected] (P.W. Kania), [email protected] (Q.Z.M. Bahlool), [email protected] (K. Buchmann). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.09.011
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1. Introduction
2.2. Parasites
Vertebrate host responses against parasitic nematodes are inadequately described and may vary considerably dependent on host and parasite species involved. In ectothermic hosts, particularly, environmental factors will also play a role. Encapsulated third stage larvae of the nematode Anisakis simplex are commonly found in a variety of tissues and organs in marine teleost fish (Smith, 1983; Abollo et al., 2001; Skov et al., 2009). Zoonotic cases occur relatively frequent and are related to ingestion of insufficiently prepared infected fish (Hochberg and Hamer, 2010). In mammals, A. simplex – and related parasitic nematodes – may elicit an immune reaction known collectively as the T helper type 2 (Th2) immune response. (Anthony et al., 2007; Patel et al., 2009). However, both mixed Th1/Th2 and Th2-driven immune responses against Anisakis larvae have been reported in rodent models (Baeza et al., 2005; Nieuwenhuizen et al., 2006) and in humans suffering from anisakiasis (del Pozo et al., 1999; Gonzalez-Muñoz et al., 2010). Bony fishes possess many of the central immune molecules and structures seen in higher vertebrates and are, like mammals, capable of mounting both an innate and adaptive immune response against invading pathogens (Whyte, 2007; Alvarez-Pellitero, 2008; Rauta et al., 2012). A fish-nematode model may therefore be useful in pinpointing pivotal elements within vertebrate host immunity to parasitic nematodes. Despite this, only a few studies have attempted to understand the immune response of fish against nematodes and the role of associated Th2-based cytokines (Buchmann, 2012), although a Th1-/Th2-like switch in rainbow trout epidermis, evoked by a parasitic protozoan, was recently demonstrated by Chettri et al. (2013). Experimental work on Anisakis-infected fish has mainly focused on basic parasitological parameters (i.e. prevalence, intensity and predilection site) but a few studies indicated that cellular responses become activated by the infection (Larsen et al., 2002; Quiazon et al., 2011; Bahlool et al., 2012). A recent immunological study suggested that injection of excretory/secretory (ES) proteins from A. simplex into rainbow trout (Oncorhynchus mykiss Walbaum) induced down-regulation of several immune relevant genes (Bahlool et al., 2013). However, no studies have yet described immune gene expression in fish following challenge with live A. simplex. It was the aim of this work to investigate the expression of 18 immune relevant genes in rainbow trout spleen and liver before and after challenge with live third stage A. simplex larvae.
Ungutted herring (Clupea harengus), freshly caught in the North Sea, were obtained from a local fishmonger. Fish were dissected and third stage larvae of A. simplex were recovered from the mesenteries and body cavity. Subsequently, isolated nematodes were transferred to a phosphate buffered saline (PBS) solution containing ampicillin (200 lg/ml) and 400 lg/ml kanamycin (Sigma–Aldrich, Denmark) for 2 h, observed through a stereomicroscope and identified to genus level. Anisakis worms were stored in a new PBS solution (without antibiotics) at 4° C for a maximum of 3 h before initiation of infection experiments. Molecular specific identification of A. simplex by PCR and subsequent sequencing of the obtained products was performed as described earlier (Bahlool et al., 2012).
2. Methods
2.3. Challenge experiment All experiments were conducted in accordance to the ethical guidelines of the Danish Ministry of Food, Agriculture and Fisheries (license: 2012-15-29300358). The oral challenges were conducted at the same time point and in duplicate. Thus, fish were kept in separate holding tanks and 140 fish were used in total; a total of 60 fish were exposed to parasites and 80 acted as controls. Individual rainbow trout were brought to an infection chamber and anesthetized with 40 mg/L tricaine-methane-sulfonate (MS222, Sigma–Aldrich, Denmark). Four A. simplex larvae were then placed in the stomach of each sedated fish using forceps. Following infection, exposed fish were transferred to an aquarium containing non-medicated water and observed for 5 min. Approximately 60% of challenged fish regurgitated worms; expelled larvae were immediately re introduced. Sham-infection was performed on control fish (forceps placed in stomach).
2.4. Parasite recovery and tissue sampling Samples comprising 10 orally challenged and 10 control fish were taken at 0, 1, 4 and 8 days post infection (d.p.i.). Fish were euthanized by an overdose of MS-222 (300 mg/L) and dissected. The liver and spleen were isolated aseptically and immediately transferred to RNAlater™ (24 h at 4° C, then stored at 20° C). Subsequently, the number of Anisakis larvae and their location was recorded from each fish. All organs and tissues were scrutinized under the microscope using glass-plate tissue compression to recover also possible larvae in organs (Buchmann, 2007).
2.1. Fish and rearing conditions 2.5. Isolation of RNA and cDNA synthesis Juvenile rainbow trout (Oncorhynchus mykiss), hatched and reared at 12 °C under pathogen-free conditions (Bornholm Salmon Hatchery, Nexø, Denmark), were transferred to the experimental fish keeping facility, University of Copenhagen, Frederiksberg. Fish (mean weight: 53.4 ± 1.2 g, mean length: 17.1 ± 0.1 cm) were acclimatized in 85 L glass aquaria for a week prior to initiation of challenge experiments. The stocking density was 25 fish per tank, water was continuously aerated and each aquarium was supplied with biofilters (Eheim, Germany). Light/dark cycle was set to 12/ 12 h and the water temperature was maintained at 14–15° C throughout the entire period. pH, ammonia (NH3), nitrate (NO3) and nitrite (NO2) concentrations were regularly tested and registered (pH 7.2, NH3 < 1.0 ppm, NO3 < 5 ppm and NO2 < 0.3 ppm) (MerckoquantÒ, Merck Chemicals, Germany). Fish were given commercial pelleted feed (Biomar, Denmark) at a rate of 1% of their biomass per day.
Homogenisation of tissue samples (40 mg of liver and 20 mg of spleen) was done by sonication on ice. RNA was extracted using GenElute™ Mammalian Total RNA kit (Sigma–Aldrich, Denmark) and removal of genomic DNA was achieved with DNase treatment (RNase free DNase I, Fermentas, Denmark). Assessment of RNA concentration (280 nm) and purity (A260/A280 ratio) was performed on a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, USA). Further, RNA product quality and integrity was confirmed by 2% ethidium bromide stained agarose gel electrophoresis. cDNA synthesis was performed with random hexamer primers in 20 ll volumes using the TaqManÒ Reverse Transcription (Applied Biosystems, USA) reagents and 800 lg template RNA. Negative control reactions lacking reverse transcriptase (RT-minus) were also carried out. Following cDNA synthesis samples were diluted 1:10 in DNase/RNase-free H2O and stored at 20° C.
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2.6. Gene expression (qPCR) Spleen and liver samples were analysed using quantitative realtime PCR (qPCR) expression of genes encoding cytokines (IL-1b, IL4/IL-13, IL-6, IL-8, IL-10, IL-22, TNFa and TGFb), cellular receptors (MHC II, CD4 and CD8) transcription factors (FoxP3a and FoxP3b), immunoglobulins (IgM and IgT), complement factor 3 (C3) and acute-phase proteins (SAA and Precerebellin). Elongation factor subunit 1-a (EF 1-a) was used as reference gene (Chettri et al., 2011). Primer and probe sequences and amplicon sizes are available as supplementary online material (S1). All qPCR assays were performed in the Stratagene Mx3005P™ real-time PCR system (AH Diagnostics as, Denmark) using the following conditions: one cycle at 95° C for 15 min, 40 cycles of a denaturation step at 95° C for 30 s and a combined annealing and elongation step at 60° C for 30 s with endpoint measurement. All wells contained a total reaction volume of 12.5 ll: 0.5 ll forward and reverse primers (10 lM each), 0.5 ll TaqManÒ probe (5 lM), 6.25 ll 2x BrilliantÒ QPCR Master Mix (AH diagnostics as, Denmark), 2.5 ll cDNA template and 2.25 ll sterile DNAse/RNAse-Free distilled water (Invitrogen, Denmark). Further, each assay included negative control wells which contained no cDNA. 2.7. Data analysis of gene expression Gene expression data from individual fish were placed into four groups: sham-infected fish (control), fish which had expelled all Anisakis at the time of sampling (worms), fish which harboured 1–4 larvae at the time of examination (+worms) and a combined group (+worms and worms pooled together). Replicate groups were tested with a two-tailed t-test (p < 0.05) and pooled if they were not significantly different. Gene expression data were analysed according to the 2 D DCt method (Livak and Schmittgen, 2001). Change in threshold cycle (DCt) was calculated as the difference between the target gene and the reference gene for each sample. DDCt was calculated as the difference between the DCt of the control group and the DCt of the experimental groups. Data are presented as the fold change of + worms group, worms group and of combined group compared to control fish. Significance was only considered when at least a 2-fold change (in order to minimize errors induced by biological variation) and a t-test result of p < 0.05 were fulfilled. 3. Results 3.1. Challenge experiment Prevalence of A. simplex larvae in challenged rainbow trout was highest 1 day post infection (d.p.i.); 75% of the fish harboured larvae (Table 1). Mean intensity and mean abundance was 1.6 ± 0.2 and 1.2 ± 0.2, respectively. Approximately half of the fish (55%) sampled 4 d.p.i. were infected, whereas a prevalence of 40% was recorded 8 d.p.i. Mean intensities of A. simplex were 1.4 ± 0.2 and 1.5 ± 0.3 at the sampling points 4 and 8 d.p.i., respectively, and corresponding mean abundances were 0.8 ± 0.2 and 0.6 ± 0.2. Larvae
Table 1 Prevalence, mean intensity and mean abundance of Anisakis simplex larvae experimentally introduced in rainbow trout. Sampling was performed 1, 4 and 8 days post infection (d.p.i.).
were primarily found between the pyloric caeca and were not visibly encapsulated. Control fish which did not receive worm larvae at day 0 were confirmed free from infection at all time points. 3.2. Gene expression Expression of 18 immune genes was investigated in rainbow trout challenged with live A. simplex larvae. Tables showing the individual gene regulations in spleen and liver of challenged fish compared to values from control fish are available online as supplementary material (S2 and S3, respectively). Basal expression levels of each gene in control fish can be seen in S4. The significantly regulated genes in spleen and liver are shown in Fig. 1 3.2.1. Gene expression – cytokines Genes encoding IL-1 (liver), IL-6 (liver), IL-22 (spleen and liver) and TNFa (liver) were generally expressed at a low level. No Ct values were obtained from the majority of liver samples with regard to the three latter genes. No cytokine genes were significantly up- or downregulated following A. simplex challenge when compared to control samples. 3.2.2. Gene expression – cellular receptors and transcription factors CD4 and CD8 genes were generally expressed at a low level in liver samples from all three experimental groups but significant up-regulation of the CD4 gene in this organ was observed in the +worms group 8 days after parasite exposure (6.1-fold change). A significant decrease of CD8 transcript levels in liver was evident in the +worms group and the combined group at 1 day post challenge (2.7 and 2.2-fold change, respectively). No significant change in regulation of the FoxP3a and FoxP3b genes were recorded. 3.2.3. Gene expression – immunoglobulins IgM gene expression was down-regulated in both investigated tissues at the sampling point 8 d.p.i.; regulation in livers from the +worms group (2.9-fold upregulation) was significantly different from control fish. A general (non-significant) down-regulation of IgT transcripts was observed in all three groups and in both organs at 8 days after parasite exposure. 3.2.4. Gene expression – C3 C3 expression was not detected in approximately 40% of spleen samples and no significant regulation was recorded in this tissue when compared to control rainbow trout. In liver, a significant up-regulation was observed in the +worms (2.2-fold change) at 4 d.p.i. 3.2.5. Gene expression – acute-phase proteins Precerebellin transcript levels were low in spleen but liver samples from fish which retained worms (+worms group) 4 d.p.i. exhibited significant up-regulations of this gene (2.2-fold change). Significant SAA gene up-regulations were found in spleen from fish in the +worms group and in the combined group at 8 days post infection (16.1 and 7.4-fold change, respectively). SAA gene expression was also higher in the liver and significant up-regulations were observed at 1 d.p.i. in the combined group (136.4-fold change) and 4 days post infection fish in the +worms, worms and combined groups (84.1, 86.4 and 85.3-fold change, respectively).
Sampling time (d.p.i.)
Prevalence (%)
Mean intensity ± SE
Mean abundance ± SE
4. Discussion
1 4 8
75 55 40
1.6 ± 0.2 1.4 ± 0.2 1.5 ± 0.3
1.2 ± 0.2 0.8 ± 0.2 0.6 ± 0.2
The present study has demonstrated that the rainbow trout/A. simplex model can be applied for elucidation of immune mechanisms in fish hosts innately resistant (repelling worms within a
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Fig. 1. Significant regulation of immune relevant genes in rainbow trout following Anisakis simplex infection. ⁄p < 0.05;
few days) and fish which are initially susceptible, but are able to activate their immunological armament and exclude worms at a reduced rate. Expression of numerous immune relevant genes differed considerably between these groups of fish. Also, gene expression levels in the combined group were not identical to values in the +worms and worms group. We therefore recommend workers focusing on the immunological interaction between vertebrates and nematodes to incorporate infection status, as presented here, into the data analysis. Some fish expelled one of the four introduced worms shortly after infection probably due to a reflex. Following re-introduction fish retained the worms at least during the observation period. It is unlikely that re-introduction of regurgitated worms had an impact on gene expression levels. It cannot be excluded that MS222 influenced the experimental fish. Elevated levels of cortisol - the stress hormone – have been reported from fish which were handled while fully anaesthetized with MS222 (Small, 2003). Increased cortisol concentrations can lead to immunosuppression in vertebrates, including fish. However, as evident from experimental studies on stressors in fish,
⁄⁄
p < 0.01.
the cortisol level is returned to equilibrium a few hours post anaesthesia (Cho and Heath, 2002; Palic´ et al., 2006). Studies on gilthead seabream (Sparus aurata L.) and rainbow trout leucocytes have found unaltered levels of respiratory burst activity, an innate immune mechanism, 1 h post MS222 anaesthesia (Ortuño et al., 2002; Kanani et al., 2013). This contradicts that MS222 would have influenced the gene expression in the present study. Further, Kanani et al. (2013) recently showed that the alternative complement response in rainbow trout is unaffected by MS222 exposure. None of the eight cytokine genes had a significant change in their level of expression in either the spleen or liver of the rainbow trout. Production of pro-inflammatory cytokines, e.g. IL-1, is stimulated upon tissue injury and up-regulation in the early phase of infection has been reported from piscine hosts responding to a variety of parasites (Lindenstrøm et al., 2003; Sigh et al., 2004; Bridle et al., 2006; Fast et al., 2006; Severin and El-Matbouli, 2007). Taking into account the tissue penetrating behaviour exhibited by A. simplex larvae, an up-regulation in expression was
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somewhat expected. The lack of cytokine gene regulation may be attributed to the general immuno-depressive effects of Anisakis E/S proteins demonstrated in a recent rainbow trout injection study (Bahlool et al., 2013). The intestinal tract of fish is equipped with gut associated lymphoid tissue (GALT) (Zapata et al., 2006) and it can be speculated that sampling from the penetration sites in the gastrointestinal system might have yielded different cytokine transcripts levels. CD4 and CD8 molecules are co-receptors located on T cells; the former is present on T helper cells whereas the latter is found on T cytotoxic lymphocytes (Randelli et al., 2008). In a recent experiment CD4 gene expression was found significantly down-regulated in rainbow trout 24 h after injection with the putative immunesupressing Anisakis E/S proteins (Bahlool et al., 2013). This was not observed in the present study where CD4 expression was significantly up-regulate-+d in the –worms group 8 days post infection. A sub-population of CD4 + cells are, together with a specific cytokine profile, associated with the Th2 type response usually seen in mammals reacting to nematodes (Buchmann, 2012). CD8 transcript levels were significantly lowered in the + worms group (and combined group) at 1 d.p.i. Immunohistochemical studies on rainbow trout infected with different parasites, such as the nematode A. simplex (see Bahlool et al., 2012) and the ciliate Ichthyophthirius multifiliis (see Olsen et al., 2011), have reported CD8 + cells to be present at the site of infection, suggesting a role of these lymphocytes in the cellular immune response. However, the constitutive expression level of both co-receptors were quite low in liver and therefore even small changes will appear as high relative fold change. Teleosts are capable of producing specific immunoglobulins towards parasitic antigens as an integrated part of the adaptive immune response (Priebe et al., 1991; Buchmann, 1993; Coscia and Oreste, 1998, 2000). The significant down-regulation of the IgM gene expression observed in the + worms group could be interpreted as a result of immune-suppression elicited by ES products from the worm as suggested by Bahlool et al. (2013). The SAA molecule is integrated in the vertebrate acute-phase response towards pathogens. In fish the acute phase response is initiated by pro-inflammatory cytokines following injury, trauma or infection and involves a variety of different innate immune components (see e.g. Alvarez-Pellitero, 2008). We did not observe any significant regulations of pro-inflammatory cytokines in this work, but the investigated acute-phase proteins showed significantly elevated gene expressions in all three groups 4 d.p.i. Live Anisakis worms have been suggested to elicit a systemic immune reaction in fish (Larsen et al., 2002) and SAA could be one factor involved. In addition to SAA, complement factor C3 gene expression was elevated in this study, corresponding to previous studies on fish reactions to parasitic invasion (Saeij et al., 2003; Jørgensen et al., 2008). This study has elucidated part of the intricate expression patterns of immune genes in bony fishes during nematode infection. It is noteworthy that the gene expression profile of rainbow trout following infection with live Anisakis simplex larvae differs from expressions in trout exposed to pure worm secretions studied by Bahlool et al. (2013). This may result from the fact that live worms also influence the host mechanically which alone would result in upregulation of a number of genes including proinflammatory cytokines (see Gonzalez et al., 2007b). When combining the action of immune-suppressive secretions and mechanical injuries the result may be a general moderation of reactions as observed in the present work. Still, genes encoding acute phase reactants including SAA and precerebellin were moderately upregulated which may reflect that these factors generally are highly activated during pathogen exposure (Gonzalez et al., 2007a). The results from our study did not point towards a clear Th2 response, as observed in certain mammalian models. Alternative pathways do,
however, most likely exist in fish (Buchmann, 2012) and the immune elements described in this work may play a role in this context. These relations should be investigated in future work on related experimental fish-nematode models. Acknowledgments This work was produced under the Danish Fish Immunology Research Centre and Network DAFINET (http://www.dafinet.dk) supported by the Danish Council for Strategic Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.exppara.2013. 09.011. References Abollo, E., Gestal, C., Pascual, S., 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87, 492–499. Alvarez-Pellitero, P., 2008. Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet. Immunol. Immunopathol. 126, 171–198. Anthony, R.M., Rutitzky, L.I., Urban, J.F., Stadecker, M.J., Gause, W.C., 2007. Protective immune mechanisms in helminth infections. Nat. Rev. Immunol. 7, 975–987. Baeza, M.L., Conejero, L., Higaki, Y., Martín, E., Pérez, C., Infante, S., Rubio, M., Zubeldia, J.M., 2005. Anisakis simplex allergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern. Clin. Exp. Immunol. 142, 433–440. Bahlool, Q.Z.M., Skovgaard, A., Kania, P.W., Haarder, S., Buchmann, K., 2013. Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol 35, 734–739. Bahlool, Q.Z.M., Skovgaard, A., Kania, P.W., Haarder, S., Buchmann, K., 2012. Microhabitat preference of Anisakis simplex in three salmonid species: immunological implications. Vet. Parasitol. 190, 489–495. Bridle, A.R., Morrison, R.N., Nowak, B.F., 2006. The expression of immuno-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease. Fish Shellfish Immunol. 20, 346–364. Buchmann, K., 1993. A note on the humoral immune response of infected Anguilla anguilla against the gill monogenean Pseudodactylogyus bini. Fish Shellfish Immunol. 3, 397–399. Buchmann, K., 2007. An Introduction to Fish Parasitological Methods. Biofolia, Frederiksberg. Buchmann, K., 2012. Fish immune responses against endoparasitic nematodes – experimental models. J. Fish Dis. 35, 623–635. Chettri, J.K., Kuhn, J.A., Jaafar, R.M., Kania, P.W., Møller, O.S., Buchmann, K., 2013. Epidermal response of rainbow trout to Ichthyobodo necator: immunohistochemical and gene expression studies indicate a Th1-/Th2-like switch. J. Fish Dis.. http://dx.doi.org/10.1111/jfd.12169. Chettri, J.K., Raida, M.K., Holten-Andersen, L., Kania, P.W., Buchmann, K., 2011. PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss). Dev. Comp. Immunol. 35, 476–482. Cho, G.K., Heath, D.D., 2002. Comparison of tricaine methensulphonate (MS222) and clove oil anaesthesia on the physiology of juvenile salmon Oncorhynchus tshawytscha (Walbaum). Aquac. Res. 31, 537–546. Coscia, M.R., Oreste, U., 1998. Presence of antibodies specific for proteins of Contracaecum osculatum (Rudolphi, 1908) in plasma of several Antarctic teleosts. Fish Shellfish Immunol. 8, 295–302. Coscia, M.R., Oreste, U., 2000. Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens. Dis. Aquat. Organ. 41, 37–42. del Pozo, V., Arrieta, I., Tuñon, T., Cortegano, I., Gomez, B., Cardaba, B., Gallardo, S., Rojo, M., Renedo, G., Palomino, P., Tabar, A.I., Lahoz, C., 1999. Immunopathogenesis of human gastrointestinal infection by Anisakis simplex. J. Allergy Clin. Immunol. 104, 637–643. Fast, M.D., Ross, N.W., Muise, D.M., Johnson, S.C., 2006. Differential gene expression in Atlantic salmon infected with Lepeophteirus salmonis. J. Aquat. Anim. Health 18, 116–127. Gonzalez-Muñoz, M., Rodriguez-Mahillo, A.I., Moneo, I., 2010. Different Th1/Th2 responses to Anisakis simplex are related to distinct clinical manifestations in sensitized patients. Parasite Immunol. 32, 67–73. Gonzalez, S.F., Buchmann, K., Nielsen, M.E., 2007a. Ichthyophthirius multifiliis infection induces massive up-regulation of serum amyloid A in carp (Cyprinus carpio). Vet. Immunol. Immunopathol. 115, 172–178. Gonzalez, S.F., Huising, M.O., Stakauskas, R., Forlenza, M., Verburg-van Kemenade, B.M.L., Buchmann, K., Nielsen, M.E., Wiegertjes, G.F., 2007b. Real-time gene
S. Haarder et al. / Experimental Parasitology 135 (2013) 564–569 expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses to injury mimicking infection with ectoparasites. Dev. Comp. Immunol. 31, 244– 254. Hochberg, N.S., Hamer, D.H., 2010. Anisakidosis: perils of the deep. Clin. Infect. Dis. 51, 806–812. Jørgensen, L., Nemli, E., Heinecke, R.D., Raida, M.K., Buchmann, K., 2008. Immunerelevant genes expressed in rainbow trout following immunisation with live vaccine against Ichthyophthirius multifiliis. Dis. Aquat. Organ. 80, 189– 197. Kanani, H.G., Soltani, M., Mirzagar, S.S., 2013. Effect of tricainemethanesulfonate (MS222), clove oil and electro-anaesthesia on respiratory burst activity in whole blood and serum alternative complement response in rainbow trout (Oncorhynchus mykiss), during the narcosis stage. Fish Shellfish Immunol. 34, 692–696. Larsen, A.H., Bresciani, J., Buchmann, K., 2002. Interactions between ecto- and endoparasites in trout Salmo trutta. Vet. Parasitol. 103, 167–173. Lindenstrøm, T., Buchmann, K., Secombes, C.J., 2003. Gyrodactylus derjavini infection elicits IL-1b expression in rainbow trout skin. Fish Shellfish Immunol. 25, 107– 115. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Nieuwenhuizen, N., Lopata, N.L., Jeebhay, M.F., Herbert, D.R., Robins, T.G., Brombacher, F., 2006. Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. J. Allergy Clin. Immunol. 117, 1098– 1105. Olsen, M.M., Kania, P.W., Heinecke, R.D., Skjoedt, K., Rasmussen, K.J., Buchmann, K., 2011. Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies. Fish Shellfish Immunol. 30, 859–869. Ortuño, J., Esteban, M.A., Meseguer, J., 2002. Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol. 12, 49–59. Palic´, D., Herold, D.M., Andreasen, C.B., Menzel, B.W., Roth, J.A., 2006. Anesthetic efficacy of tricaine methanesulfonate, metomidate and eugenol: effects on plasma cortisol concentration and neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820). Aquaculture 254, 675–685.
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Patel, N., Kreider, T., Urban, J.F., Gause, W.C., 2009. Characterisation of effector mechanisms at the host:parasite interface during the immune response to tissue-dwelling intestinal nematode parasites. Int. J. Parasitol. 39, 13–21. Priebe, K., Huber, C., Märtlbauer, E., Terplan, G., 1991. Detection of antibodies against the larva of Anisakis simplex in the pollock Pollachius virens using ELISA. Zbl. Vet. Med. B 38, 209–214. Quiazon, K.M., Yoshinaga, T., Ogawa, K., 2011. Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder. Parasitol. Int. 60, 126–131. Randelli, E., Buonocore, F., Scapigliati, G., 2008. Cell markers and determinants in fish immunology. Fish Shellfish Immunol. 25, 326–340. Rauta, P.R., Nayak, B., Das, S., 2012. Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunol. Lett. 148, 23–33. Saeij, J.P.J., de Vries, B.J., Wiegertjes, G.F., 2003. The immune response of carp to Trypanosoma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation. Dev. Comp. Immunol. 27, 859–874. Severin, V.I.C., El-Matbouli, M., 2007. Relative quantification of immune-regulatory genes in two rainbow trout strains, Oncorhynchus mykiss, after exposure to Myxobolus cerebralis, the causative agent of whirling disease. Parasitol. Res. 101, 1019–1027. Sigh, J., Lindenstrøm, T., Buchmann, K., 2004. Expression of pro-inflammatory cytokines in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multifiliis. Fish Shellfish Immunol. 17, 75–86. Skov, J., Kania, P.W., Olsen, M.M., Lauridsen, J.H., Buchmann, K., 2009. Nematode infections of maricultured and wild fishes in Danish waters: a comparative study. Aquaculture 298, 24–28. Small, C.B., 2003. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulphonate, quinaldine and clove oil anesthesized channel catfish Ictalurus punctatus. Aquaculture 218, 177–185. Smith, J.W., 1983. Anisakis simplex (Rudolphi, 1809, det. Krabbe, 1878) (Nematoda: Ascaridoidea): morphology and morphometry of larvae from euphausiids and fish, and a review of the life-history and ecology. J. Helminthol. 57, 205–224. Whyte, S.K., 2007. The innate immune response of finfish – a review of current knowledge. Fish Shellfish Immunol. 23, 1127–1151. Zapata, A., Diez, B., Cejalvo, T., Gutierrez-de Frias, C., Cortes, A., 2006. Ontogeny of the immune system of fish. Fish Shellfish Immunol. 20, 126–136.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae Simon Haarder ⇑, Per W. Kania, Qusay Z.M. Bahlool, Kurt Buchmann Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 7, Frederiksberg C, Denmark
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Oral challenge with live Anisakis
simplex induces expression of numerous immune relevant genes in rainbow trout. The CD8 and IgM genes were significantly down-regulated in infected fish at 1 and 8 days post infection (d.p.i.). A significant up-regulation was seen in the CD4 gene in fish which rejected the worms at 8 d.p.i. C3 and precerebellin genes were both significantly up-regulated in infected fish 4 d.p.i. The SAA gene exhibited significant up-regulation at several different sampling points.
a r t i c l e
i n f o
Article history: Received 31 January 2013 Received in revised form 19 August 2013 Accepted 6 September 2013 Available online 16 September 2013 Keywords: Anisakis simplex Fish immunology Gene expression Fish-nematode experimental model qPCR Rainbow trout
a b s t r a c t Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fishnematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8 days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; ‘‘worms rejected’’ (worms), ‘‘worms present’’ (+worms) and a combined group consisting of samples pooled from both previous groups (/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8 d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24 h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8 d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4 d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae. Ó 2013 Elsevier Inc. All rights reserved.
⇑ Corresponding author. E-mail addresses: [email protected] (S. Haarder), [email protected] (P.W. Kania), [email protected] (Q.Z.M. Bahlool), [email protected] (K. Buchmann). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.09.011
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1. Introduction
2.2. Parasites
Vertebrate host responses against parasitic nematodes are inadequately described and may vary considerably dependent on host and parasite species involved. In ectothermic hosts, particularly, environmental factors will also play a role. Encapsulated third stage larvae of the nematode Anisakis simplex are commonly found in a variety of tissues and organs in marine teleost fish (Smith, 1983; Abollo et al., 2001; Skov et al., 2009). Zoonotic cases occur relatively frequent and are related to ingestion of insufficiently prepared infected fish (Hochberg and Hamer, 2010). In mammals, A. simplex – and related parasitic nematodes – may elicit an immune reaction known collectively as the T helper type 2 (Th2) immune response. (Anthony et al., 2007; Patel et al., 2009). However, both mixed Th1/Th2 and Th2-driven immune responses against Anisakis larvae have been reported in rodent models (Baeza et al., 2005; Nieuwenhuizen et al., 2006) and in humans suffering from anisakiasis (del Pozo et al., 1999; Gonzalez-Muñoz et al., 2010). Bony fishes possess many of the central immune molecules and structures seen in higher vertebrates and are, like mammals, capable of mounting both an innate and adaptive immune response against invading pathogens (Whyte, 2007; Alvarez-Pellitero, 2008; Rauta et al., 2012). A fish-nematode model may therefore be useful in pinpointing pivotal elements within vertebrate host immunity to parasitic nematodes. Despite this, only a few studies have attempted to understand the immune response of fish against nematodes and the role of associated Th2-based cytokines (Buchmann, 2012), although a Th1-/Th2-like switch in rainbow trout epidermis, evoked by a parasitic protozoan, was recently demonstrated by Chettri et al. (2013). Experimental work on Anisakis-infected fish has mainly focused on basic parasitological parameters (i.e. prevalence, intensity and predilection site) but a few studies indicated that cellular responses become activated by the infection (Larsen et al., 2002; Quiazon et al., 2011; Bahlool et al., 2012). A recent immunological study suggested that injection of excretory/secretory (ES) proteins from A. simplex into rainbow trout (Oncorhynchus mykiss Walbaum) induced down-regulation of several immune relevant genes (Bahlool et al., 2013). However, no studies have yet described immune gene expression in fish following challenge with live A. simplex. It was the aim of this work to investigate the expression of 18 immune relevant genes in rainbow trout spleen and liver before and after challenge with live third stage A. simplex larvae.
Ungutted herring (Clupea harengus), freshly caught in the North Sea, were obtained from a local fishmonger. Fish were dissected and third stage larvae of A. simplex were recovered from the mesenteries and body cavity. Subsequently, isolated nematodes were transferred to a phosphate buffered saline (PBS) solution containing ampicillin (200 lg/ml) and 400 lg/ml kanamycin (Sigma–Aldrich, Denmark) for 2 h, observed through a stereomicroscope and identified to genus level. Anisakis worms were stored in a new PBS solution (without antibiotics) at 4° C for a maximum of 3 h before initiation of infection experiments. Molecular specific identification of A. simplex by PCR and subsequent sequencing of the obtained products was performed as described earlier (Bahlool et al., 2012).
2. Methods
2.3. Challenge experiment All experiments were conducted in accordance to the ethical guidelines of the Danish Ministry of Food, Agriculture and Fisheries (license: 2012-15-29300358). The oral challenges were conducted at the same time point and in duplicate. Thus, fish were kept in separate holding tanks and 140 fish were used in total; a total of 60 fish were exposed to parasites and 80 acted as controls. Individual rainbow trout were brought to an infection chamber and anesthetized with 40 mg/L tricaine-methane-sulfonate (MS222, Sigma–Aldrich, Denmark). Four A. simplex larvae were then placed in the stomach of each sedated fish using forceps. Following infection, exposed fish were transferred to an aquarium containing non-medicated water and observed for 5 min. Approximately 60% of challenged fish regurgitated worms; expelled larvae were immediately re introduced. Sham-infection was performed on control fish (forceps placed in stomach).
2.4. Parasite recovery and tissue sampling Samples comprising 10 orally challenged and 10 control fish were taken at 0, 1, 4 and 8 days post infection (d.p.i.). Fish were euthanized by an overdose of MS-222 (300 mg/L) and dissected. The liver and spleen were isolated aseptically and immediately transferred to RNAlater™ (24 h at 4° C, then stored at 20° C). Subsequently, the number of Anisakis larvae and their location was recorded from each fish. All organs and tissues were scrutinized under the microscope using glass-plate tissue compression to recover also possible larvae in organs (Buchmann, 2007).
2.1. Fish and rearing conditions 2.5. Isolation of RNA and cDNA synthesis Juvenile rainbow trout (Oncorhynchus mykiss), hatched and reared at 12 °C under pathogen-free conditions (Bornholm Salmon Hatchery, Nexø, Denmark), were transferred to the experimental fish keeping facility, University of Copenhagen, Frederiksberg. Fish (mean weight: 53.4 ± 1.2 g, mean length: 17.1 ± 0.1 cm) were acclimatized in 85 L glass aquaria for a week prior to initiation of challenge experiments. The stocking density was 25 fish per tank, water was continuously aerated and each aquarium was supplied with biofilters (Eheim, Germany). Light/dark cycle was set to 12/ 12 h and the water temperature was maintained at 14–15° C throughout the entire period. pH, ammonia (NH3), nitrate (NO3) and nitrite (NO2) concentrations were regularly tested and registered (pH 7.2, NH3 < 1.0 ppm, NO3 < 5 ppm and NO2 < 0.3 ppm) (MerckoquantÒ, Merck Chemicals, Germany). Fish were given commercial pelleted feed (Biomar, Denmark) at a rate of 1% of their biomass per day.
Homogenisation of tissue samples (40 mg of liver and 20 mg of spleen) was done by sonication on ice. RNA was extracted using GenElute™ Mammalian Total RNA kit (Sigma–Aldrich, Denmark) and removal of genomic DNA was achieved with DNase treatment (RNase free DNase I, Fermentas, Denmark). Assessment of RNA concentration (280 nm) and purity (A260/A280 ratio) was performed on a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, USA). Further, RNA product quality and integrity was confirmed by 2% ethidium bromide stained agarose gel electrophoresis. cDNA synthesis was performed with random hexamer primers in 20 ll volumes using the TaqManÒ Reverse Transcription (Applied Biosystems, USA) reagents and 800 lg template RNA. Negative control reactions lacking reverse transcriptase (RT-minus) were also carried out. Following cDNA synthesis samples were diluted 1:10 in DNase/RNase-free H2O and stored at 20° C.
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2.6. Gene expression (qPCR) Spleen and liver samples were analysed using quantitative realtime PCR (qPCR) expression of genes encoding cytokines (IL-1b, IL4/IL-13, IL-6, IL-8, IL-10, IL-22, TNFa and TGFb), cellular receptors (MHC II, CD4 and CD8) transcription factors (FoxP3a and FoxP3b), immunoglobulins (IgM and IgT), complement factor 3 (C3) and acute-phase proteins (SAA and Precerebellin). Elongation factor subunit 1-a (EF 1-a) was used as reference gene (Chettri et al., 2011). Primer and probe sequences and amplicon sizes are available as supplementary online material (S1). All qPCR assays were performed in the Stratagene Mx3005P™ real-time PCR system (AH Diagnostics as, Denmark) using the following conditions: one cycle at 95° C for 15 min, 40 cycles of a denaturation step at 95° C for 30 s and a combined annealing and elongation step at 60° C for 30 s with endpoint measurement. All wells contained a total reaction volume of 12.5 ll: 0.5 ll forward and reverse primers (10 lM each), 0.5 ll TaqManÒ probe (5 lM), 6.25 ll 2x BrilliantÒ QPCR Master Mix (AH diagnostics as, Denmark), 2.5 ll cDNA template and 2.25 ll sterile DNAse/RNAse-Free distilled water (Invitrogen, Denmark). Further, each assay included negative control wells which contained no cDNA. 2.7. Data analysis of gene expression Gene expression data from individual fish were placed into four groups: sham-infected fish (control), fish which had expelled all Anisakis at the time of sampling (worms), fish which harboured 1–4 larvae at the time of examination (+worms) and a combined group (+worms and worms pooled together). Replicate groups were tested with a two-tailed t-test (p < 0.05) and pooled if they were not significantly different. Gene expression data were analysed according to the 2 D DCt method (Livak and Schmittgen, 2001). Change in threshold cycle (DCt) was calculated as the difference between the target gene and the reference gene for each sample. DDCt was calculated as the difference between the DCt of the control group and the DCt of the experimental groups. Data are presented as the fold change of + worms group, worms group and of combined group compared to control fish. Significance was only considered when at least a 2-fold change (in order to minimize errors induced by biological variation) and a t-test result of p < 0.05 were fulfilled. 3. Results 3.1. Challenge experiment Prevalence of A. simplex larvae in challenged rainbow trout was highest 1 day post infection (d.p.i.); 75% of the fish harboured larvae (Table 1). Mean intensity and mean abundance was 1.6 ± 0.2 and 1.2 ± 0.2, respectively. Approximately half of the fish (55%) sampled 4 d.p.i. were infected, whereas a prevalence of 40% was recorded 8 d.p.i. Mean intensities of A. simplex were 1.4 ± 0.2 and 1.5 ± 0.3 at the sampling points 4 and 8 d.p.i., respectively, and corresponding mean abundances were 0.8 ± 0.2 and 0.6 ± 0.2. Larvae
Table 1 Prevalence, mean intensity and mean abundance of Anisakis simplex larvae experimentally introduced in rainbow trout. Sampling was performed 1, 4 and 8 days post infection (d.p.i.).
were primarily found between the pyloric caeca and were not visibly encapsulated. Control fish which did not receive worm larvae at day 0 were confirmed free from infection at all time points. 3.2. Gene expression Expression of 18 immune genes was investigated in rainbow trout challenged with live A. simplex larvae. Tables showing the individual gene regulations in spleen and liver of challenged fish compared to values from control fish are available online as supplementary material (S2 and S3, respectively). Basal expression levels of each gene in control fish can be seen in S4. The significantly regulated genes in spleen and liver are shown in Fig. 1 3.2.1. Gene expression – cytokines Genes encoding IL-1 (liver), IL-6 (liver), IL-22 (spleen and liver) and TNFa (liver) were generally expressed at a low level. No Ct values were obtained from the majority of liver samples with regard to the three latter genes. No cytokine genes were significantly up- or downregulated following A. simplex challenge when compared to control samples. 3.2.2. Gene expression – cellular receptors and transcription factors CD4 and CD8 genes were generally expressed at a low level in liver samples from all three experimental groups but significant up-regulation of the CD4 gene in this organ was observed in the +worms group 8 days after parasite exposure (6.1-fold change). A significant decrease of CD8 transcript levels in liver was evident in the +worms group and the combined group at 1 day post challenge (2.7 and 2.2-fold change, respectively). No significant change in regulation of the FoxP3a and FoxP3b genes were recorded. 3.2.3. Gene expression – immunoglobulins IgM gene expression was down-regulated in both investigated tissues at the sampling point 8 d.p.i.; regulation in livers from the +worms group (2.9-fold upregulation) was significantly different from control fish. A general (non-significant) down-regulation of IgT transcripts was observed in all three groups and in both organs at 8 days after parasite exposure. 3.2.4. Gene expression – C3 C3 expression was not detected in approximately 40% of spleen samples and no significant regulation was recorded in this tissue when compared to control rainbow trout. In liver, a significant up-regulation was observed in the +worms (2.2-fold change) at 4 d.p.i. 3.2.5. Gene expression – acute-phase proteins Precerebellin transcript levels were low in spleen but liver samples from fish which retained worms (+worms group) 4 d.p.i. exhibited significant up-regulations of this gene (2.2-fold change). Significant SAA gene up-regulations were found in spleen from fish in the +worms group and in the combined group at 8 days post infection (16.1 and 7.4-fold change, respectively). SAA gene expression was also higher in the liver and significant up-regulations were observed at 1 d.p.i. in the combined group (136.4-fold change) and 4 days post infection fish in the +worms, worms and combined groups (84.1, 86.4 and 85.3-fold change, respectively).
Sampling time (d.p.i.)
Prevalence (%)
Mean intensity ± SE
Mean abundance ± SE
4. Discussion
1 4 8
75 55 40
1.6 ± 0.2 1.4 ± 0.2 1.5 ± 0.3
1.2 ± 0.2 0.8 ± 0.2 0.6 ± 0.2
The present study has demonstrated that the rainbow trout/A. simplex model can be applied for elucidation of immune mechanisms in fish hosts innately resistant (repelling worms within a
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Fig. 1. Significant regulation of immune relevant genes in rainbow trout following Anisakis simplex infection. ⁄p < 0.05;
few days) and fish which are initially susceptible, but are able to activate their immunological armament and exclude worms at a reduced rate. Expression of numerous immune relevant genes differed considerably between these groups of fish. Also, gene expression levels in the combined group were not identical to values in the +worms and worms group. We therefore recommend workers focusing on the immunological interaction between vertebrates and nematodes to incorporate infection status, as presented here, into the data analysis. Some fish expelled one of the four introduced worms shortly after infection probably due to a reflex. Following re-introduction fish retained the worms at least during the observation period. It is unlikely that re-introduction of regurgitated worms had an impact on gene expression levels. It cannot be excluded that MS222 influenced the experimental fish. Elevated levels of cortisol - the stress hormone – have been reported from fish which were handled while fully anaesthetized with MS222 (Small, 2003). Increased cortisol concentrations can lead to immunosuppression in vertebrates, including fish. However, as evident from experimental studies on stressors in fish,
⁄⁄
p < 0.01.
the cortisol level is returned to equilibrium a few hours post anaesthesia (Cho and Heath, 2002; Palic´ et al., 2006). Studies on gilthead seabream (Sparus aurata L.) and rainbow trout leucocytes have found unaltered levels of respiratory burst activity, an innate immune mechanism, 1 h post MS222 anaesthesia (Ortuño et al., 2002; Kanani et al., 2013). This contradicts that MS222 would have influenced the gene expression in the present study. Further, Kanani et al. (2013) recently showed that the alternative complement response in rainbow trout is unaffected by MS222 exposure. None of the eight cytokine genes had a significant change in their level of expression in either the spleen or liver of the rainbow trout. Production of pro-inflammatory cytokines, e.g. IL-1, is stimulated upon tissue injury and up-regulation in the early phase of infection has been reported from piscine hosts responding to a variety of parasites (Lindenstrøm et al., 2003; Sigh et al., 2004; Bridle et al., 2006; Fast et al., 2006; Severin and El-Matbouli, 2007). Taking into account the tissue penetrating behaviour exhibited by A. simplex larvae, an up-regulation in expression was
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somewhat expected. The lack of cytokine gene regulation may be attributed to the general immuno-depressive effects of Anisakis E/S proteins demonstrated in a recent rainbow trout injection study (Bahlool et al., 2013). The intestinal tract of fish is equipped with gut associated lymphoid tissue (GALT) (Zapata et al., 2006) and it can be speculated that sampling from the penetration sites in the gastrointestinal system might have yielded different cytokine transcripts levels. CD4 and CD8 molecules are co-receptors located on T cells; the former is present on T helper cells whereas the latter is found on T cytotoxic lymphocytes (Randelli et al., 2008). In a recent experiment CD4 gene expression was found significantly down-regulated in rainbow trout 24 h after injection with the putative immunesupressing Anisakis E/S proteins (Bahlool et al., 2013). This was not observed in the present study where CD4 expression was significantly up-regulate-+d in the –worms group 8 days post infection. A sub-population of CD4 + cells are, together with a specific cytokine profile, associated with the Th2 type response usually seen in mammals reacting to nematodes (Buchmann, 2012). CD8 transcript levels were significantly lowered in the + worms group (and combined group) at 1 d.p.i. Immunohistochemical studies on rainbow trout infected with different parasites, such as the nematode A. simplex (see Bahlool et al., 2012) and the ciliate Ichthyophthirius multifiliis (see Olsen et al., 2011), have reported CD8 + cells to be present at the site of infection, suggesting a role of these lymphocytes in the cellular immune response. However, the constitutive expression level of both co-receptors were quite low in liver and therefore even small changes will appear as high relative fold change. Teleosts are capable of producing specific immunoglobulins towards parasitic antigens as an integrated part of the adaptive immune response (Priebe et al., 1991; Buchmann, 1993; Coscia and Oreste, 1998, 2000). The significant down-regulation of the IgM gene expression observed in the + worms group could be interpreted as a result of immune-suppression elicited by ES products from the worm as suggested by Bahlool et al. (2013). The SAA molecule is integrated in the vertebrate acute-phase response towards pathogens. In fish the acute phase response is initiated by pro-inflammatory cytokines following injury, trauma or infection and involves a variety of different innate immune components (see e.g. Alvarez-Pellitero, 2008). We did not observe any significant regulations of pro-inflammatory cytokines in this work, but the investigated acute-phase proteins showed significantly elevated gene expressions in all three groups 4 d.p.i. Live Anisakis worms have been suggested to elicit a systemic immune reaction in fish (Larsen et al., 2002) and SAA could be one factor involved. In addition to SAA, complement factor C3 gene expression was elevated in this study, corresponding to previous studies on fish reactions to parasitic invasion (Saeij et al., 2003; Jørgensen et al., 2008). This study has elucidated part of the intricate expression patterns of immune genes in bony fishes during nematode infection. It is noteworthy that the gene expression profile of rainbow trout following infection with live Anisakis simplex larvae differs from expressions in trout exposed to pure worm secretions studied by Bahlool et al. (2013). This may result from the fact that live worms also influence the host mechanically which alone would result in upregulation of a number of genes including proinflammatory cytokines (see Gonzalez et al., 2007b). When combining the action of immune-suppressive secretions and mechanical injuries the result may be a general moderation of reactions as observed in the present work. Still, genes encoding acute phase reactants including SAA and precerebellin were moderately upregulated which may reflect that these factors generally are highly activated during pathogen exposure (Gonzalez et al., 2007a). The results from our study did not point towards a clear Th2 response, as observed in certain mammalian models. Alternative pathways do,
however, most likely exist in fish (Buchmann, 2012) and the immune elements described in this work may play a role in this context. These relations should be investigated in future work on related experimental fish-nematode models. Acknowledgments This work was produced under the Danish Fish Immunology Research Centre and Network DAFINET (http://www.dafinet.dk) supported by the Danish Council for Strategic Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.exppara.2013. 09.011. References Abollo, E., Gestal, C., Pascual, S., 2001. Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87, 492–499. Alvarez-Pellitero, P., 2008. Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet. Immunol. Immunopathol. 126, 171–198. Anthony, R.M., Rutitzky, L.I., Urban, J.F., Stadecker, M.J., Gause, W.C., 2007. Protective immune mechanisms in helminth infections. Nat. Rev. Immunol. 7, 975–987. Baeza, M.L., Conejero, L., Higaki, Y., Martín, E., Pérez, C., Infante, S., Rubio, M., Zubeldia, J.M., 2005. Anisakis simplex allergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern. Clin. Exp. Immunol. 142, 433–440. Bahlool, Q.Z.M., Skovgaard, A., Kania, P.W., Haarder, S., Buchmann, K., 2013. Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol 35, 734–739. Bahlool, Q.Z.M., Skovgaard, A., Kania, P.W., Haarder, S., Buchmann, K., 2012. Microhabitat preference of Anisakis simplex in three salmonid species: immunological implications. Vet. Parasitol. 190, 489–495. Bridle, A.R., Morrison, R.N., Nowak, B.F., 2006. The expression of immuno-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease. Fish Shellfish Immunol. 20, 346–364. Buchmann, K., 1993. A note on the humoral immune response of infected Anguilla anguilla against the gill monogenean Pseudodactylogyus bini. Fish Shellfish Immunol. 3, 397–399. Buchmann, K., 2007. An Introduction to Fish Parasitological Methods. Biofolia, Frederiksberg. Buchmann, K., 2012. Fish immune responses against endoparasitic nematodes – experimental models. J. Fish Dis. 35, 623–635. Chettri, J.K., Kuhn, J.A., Jaafar, R.M., Kania, P.W., Møller, O.S., Buchmann, K., 2013. Epidermal response of rainbow trout to Ichthyobodo necator: immunohistochemical and gene expression studies indicate a Th1-/Th2-like switch. J. Fish Dis.. http://dx.doi.org/10.1111/jfd.12169. Chettri, J.K., Raida, M.K., Holten-Andersen, L., Kania, P.W., Buchmann, K., 2011. PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss). Dev. Comp. Immunol. 35, 476–482. Cho, G.K., Heath, D.D., 2002. Comparison of tricaine methensulphonate (MS222) and clove oil anaesthesia on the physiology of juvenile salmon Oncorhynchus tshawytscha (Walbaum). Aquac. Res. 31, 537–546. Coscia, M.R., Oreste, U., 1998. Presence of antibodies specific for proteins of Contracaecum osculatum (Rudolphi, 1908) in plasma of several Antarctic teleosts. Fish Shellfish Immunol. 8, 295–302. Coscia, M.R., Oreste, U., 2000. Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens. Dis. Aquat. Organ. 41, 37–42. del Pozo, V., Arrieta, I., Tuñon, T., Cortegano, I., Gomez, B., Cardaba, B., Gallardo, S., Rojo, M., Renedo, G., Palomino, P., Tabar, A.I., Lahoz, C., 1999. Immunopathogenesis of human gastrointestinal infection by Anisakis simplex. J. Allergy Clin. Immunol. 104, 637–643. Fast, M.D., Ross, N.W., Muise, D.M., Johnson, S.C., 2006. Differential gene expression in Atlantic salmon infected with Lepeophteirus salmonis. J. Aquat. Anim. Health 18, 116–127. Gonzalez-Muñoz, M., Rodriguez-Mahillo, A.I., Moneo, I., 2010. Different Th1/Th2 responses to Anisakis simplex are related to distinct clinical manifestations in sensitized patients. Parasite Immunol. 32, 67–73. Gonzalez, S.F., Buchmann, K., Nielsen, M.E., 2007a. Ichthyophthirius multifiliis infection induces massive up-regulation of serum amyloid A in carp (Cyprinus carpio). Vet. Immunol. Immunopathol. 115, 172–178. Gonzalez, S.F., Huising, M.O., Stakauskas, R., Forlenza, M., Verburg-van Kemenade, B.M.L., Buchmann, K., Nielsen, M.E., Wiegertjes, G.F., 2007b. Real-time gene
S. Haarder et al. / Experimental Parasitology 135 (2013) 564–569 expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses to injury mimicking infection with ectoparasites. Dev. Comp. Immunol. 31, 244– 254. Hochberg, N.S., Hamer, D.H., 2010. Anisakidosis: perils of the deep. Clin. Infect. Dis. 51, 806–812. Jørgensen, L., Nemli, E., Heinecke, R.D., Raida, M.K., Buchmann, K., 2008. Immunerelevant genes expressed in rainbow trout following immunisation with live vaccine against Ichthyophthirius multifiliis. Dis. Aquat. Organ. 80, 189– 197. Kanani, H.G., Soltani, M., Mirzagar, S.S., 2013. Effect of tricainemethanesulfonate (MS222), clove oil and electro-anaesthesia on respiratory burst activity in whole blood and serum alternative complement response in rainbow trout (Oncorhynchus mykiss), during the narcosis stage. Fish Shellfish Immunol. 34, 692–696. Larsen, A.H., Bresciani, J., Buchmann, K., 2002. Interactions between ecto- and endoparasites in trout Salmo trutta. Vet. Parasitol. 103, 167–173. Lindenstrøm, T., Buchmann, K., Secombes, C.J., 2003. Gyrodactylus derjavini infection elicits IL-1b expression in rainbow trout skin. Fish Shellfish Immunol. 25, 107– 115. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Nieuwenhuizen, N., Lopata, N.L., Jeebhay, M.F., Herbert, D.R., Robins, T.G., Brombacher, F., 2006. Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. J. Allergy Clin. Immunol. 117, 1098– 1105. Olsen, M.M., Kania, P.W., Heinecke, R.D., Skjoedt, K., Rasmussen, K.J., Buchmann, K., 2011. Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies. Fish Shellfish Immunol. 30, 859–869. Ortuño, J., Esteban, M.A., Meseguer, J., 2002. Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol. 12, 49–59. Palic´, D., Herold, D.M., Andreasen, C.B., Menzel, B.W., Roth, J.A., 2006. Anesthetic efficacy of tricaine methanesulfonate, metomidate and eugenol: effects on plasma cortisol concentration and neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820). Aquaculture 254, 675–685.
569
Patel, N., Kreider, T., Urban, J.F., Gause, W.C., 2009. Characterisation of effector mechanisms at the host:parasite interface during the immune response to tissue-dwelling intestinal nematode parasites. Int. J. Parasitol. 39, 13–21. Priebe, K., Huber, C., Märtlbauer, E., Terplan, G., 1991. Detection of antibodies against the larva of Anisakis simplex in the pollock Pollachius virens using ELISA. Zbl. Vet. Med. B 38, 209–214. Quiazon, K.M., Yoshinaga, T., Ogawa, K., 2011. Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder. Parasitol. Int. 60, 126–131. Randelli, E., Buonocore, F., Scapigliati, G., 2008. Cell markers and determinants in fish immunology. Fish Shellfish Immunol. 25, 326–340. Rauta, P.R., Nayak, B., Das, S., 2012. Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunol. Lett. 148, 23–33. Saeij, J.P.J., de Vries, B.J., Wiegertjes, G.F., 2003. The immune response of carp to Trypanosoma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation. Dev. Comp. Immunol. 27, 859–874. Severin, V.I.C., El-Matbouli, M., 2007. Relative quantification of immune-regulatory genes in two rainbow trout strains, Oncorhynchus mykiss, after exposure to Myxobolus cerebralis, the causative agent of whirling disease. Parasitol. Res. 101, 1019–1027. Sigh, J., Lindenstrøm, T., Buchmann, K., 2004. Expression of pro-inflammatory cytokines in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multifiliis. Fish Shellfish Immunol. 17, 75–86. Skov, J., Kania, P.W., Olsen, M.M., Lauridsen, J.H., Buchmann, K., 2009. Nematode infections of maricultured and wild fishes in Danish waters: a comparative study. Aquaculture 298, 24–28. Small, C.B., 2003. Anesthetic efficacy of metomidate and comparison of plasma cortisol responses to tricaine methanesulphonate, quinaldine and clove oil anesthesized channel catfish Ictalurus punctatus. Aquaculture 218, 177–185. Smith, J.W., 1983. Anisakis simplex (Rudolphi, 1809, det. Krabbe, 1878) (Nematoda: Ascaridoidea): morphology and morphometry of larvae from euphausiids and fish, and a review of the life-history and ecology. J. Helminthol. 57, 205–224. Whyte, S.K., 2007. The innate immune response of finfish – a review of current knowledge. Fish Shellfish Immunol. 23, 1127–1151. Zapata, A., Diez, B., Cejalvo, T., Gutierrez-de Frias, C., Cortes, A., 2006. Ontogeny of the immune system of fish. Fish Shellfish Immunol. 20, 126–136.